Apr 08, 2025

Public workspaceDifferential memory enrichment of cytotoxic CD4 T cells in Parkinson’s disease patients reactive to ⍺-synuclein - Cytotoxic panel staining

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm9bn9l3p/v1
  • Antoine Freuchet1,
  • Emil Johansson1,
  • cecilia 1,
  • Alessandro Sette1
  • 1La Jolla Institute for Immunology
Emil Johansson
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm9bn9l3p/v1
Protocol Citation: Antoine Freuchet, Emil Johansson, cecilia , Alessandro Sette 2025. Differential memory enrichment of cytotoxic CD4 T cells in Parkinson’s disease patients reactive to ⍺-synuclein - Cytotoxic panel staining. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9bn9l3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 10, 2025
Last Modified: April 08, 2025
Protocol Integer ID: 119912
Keywords: parkinson, differential memory enrichment, cytotoxic panel
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000375
Abstract
The purpose of this protocol is to quantify the number of cytotoxic CD4 T cells in the different memory compartments
Troubleshooting
Prepare FACS buffer
FACS buffer recipe (2mM EDTA, 2%FBS in PBS)
  1. 500ml PBS pH 7.4 Gibco
  2. 10 ml FBS
  3. 2ml 0.5 M EDTA
  4. Filter through 0.2µm filter and de-gas for 30 minutes
  5. Store at 4˚C
Setup
  1. Thaw indicated number of vial(s) of PBMC.
  2. Place the vials in 37C water bath for 1 min. Transfer to sterile 15 ml Falcon Tubes with 10 mL HR5 and 20 µL benzonase.
  3. Centrifuge @ 1200 rpm (9/9 accel/break) for 7 min @ 4C.
  4. Resuspend cells in 10 ml HR5 and count cell number.
  5. Centrifuge cells @ 1200 rpm for 7 min @ 4C.
  6. Resuspend HR5 medium to get a final concentration of 20x106 cells/ml.
  7. Seed 2x100 µl of the 20x106 cells/ml cell suspension into 96U well plate (20^6 cells/100uL) for each condition for the antibody staining.
  8. For this experiment, seed two wells per donor for cells that will be stimulated with PMA/Ionomycin, and two wells that will be left unstimulated.
  9. Add PMA and Ionomycin to reach a final concentration of 1μg/mL to the wells designated for stimulated cells.
  10. Add GolgiPlug and GolgiStop to all wells, to reach a final volume of 2μg/mL. Incubate cells for 4h at 37°C 5% CO2.
Staining
Antibody staining

  1. Centrifuge cells at 1400 rpm/5 min/4C.
  2. Wash with 200 uL PBS per well and cfg at 1400 rpm/5 min/4C. ]
  3. Add 100 µl/pts of Live/Dead eF506 (1/1000 in PBS) and Fc Block (1/80 in PBS). Incubate 20 min in dark at 4C.

ReagentFlurochromeAmount per well (100 uL total)
Viability DyeeF5060.1
Fc Block-1.25
PBS-98.65

  1. Add 100uL of FACS Buffer/well. Centrifuge cells at 1400 rpm/5 min/4C.
  2. Add 100uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  3. Resuspend cells in 100 uL of antibody mix and incubate for 30 min at 4C.


Antibody targetFlurochromeAmount per well (100 uL total)
CD4BUV3952.50
CD8BV6502.50
CCR7AF4882.50
CD45RABV5702.50
CD16BUV7372.50
CD56BV4212.50
CD94PerCPCy5.52.50
Brillian Stain Buffer Plus-10
FACS Buffer-72.5


  1. Add 100uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  2. Add 200uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  3. Add 200uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  4. Fix and permeabilize cells by resuspending cells in 100 µl Fixation/Permeabilization working solution from the Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher). Incubate cells for 30 minutes in the dark at room temperature (RT).
  5. Resuspend cells in Intracellular antibody cocktail:
Antibody targetFlurochromeAmount per well (100 µl per well)
CD3AF7002.50
PerforinPE-Cy72.50
GzmBPE-CF5942.50
GranulysinPE2.50
CCL4APC2.50
Brilliant Stain Buffer Plus-10
1X Permeabilization Buffer-77.5

  1. Incubate cells for 45 minutes at RT in the dark.
  2. Add 100uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  3. Add 200uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  4. Add 200uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  5. Add 100 uL 4% PFA to each well. Incubate cells for 20 minutes in the dark at 4C.
  6. Add 100uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  7. Add 200uL of FACS Buffer /well. Centrifuge cells at 1400 rpm/5 min/4C.
  8. Resuspend in 150 µL of FACS Buffer /well.
  9. Ready to acquire on flow cytometer